Two-photon microscopy as a tool to investigate the therapeutic time window of methylprednisolone in a mouse spinal cord injury model.

Purpose: The aim of the present study was to explore the use of two-photon microscopy for investigating the therapeutic time window of methylprednisolone (MP) treatment after spinal cord injury (SCI). Methods: Twenty-four YFP H-line mice were subjected to hemisection SCI and then divided into four groups. Group 1 received MP at 30 min post-injury; group 2 received MP at 8 h post-injury; group 3 received MP at 24 h post-injury; and group 4 received saline at 30 min post-injury. Post-injury axonal dieback was imaged in vivo using two-photon microscopy. After all imaging sessions, histological examination of the surviving neurons and microglial/macrophage accumulation was performed. Results: Two-photon imaging revealed the degree of progressive axon damage after SCI. Group 1 exhibited a shorter axonal dieback distance and slower axonal dieback speed than groups 2, 3, and 4 (p < 0.01). MAP-2 staining revealed greater neuronal survival in group 1 than in groups 2, 3, and 4 (p < 0.05). F4/80 staining revealed greater microglial/macrophage density in groups 2, 3, and 4 than in group 1 (p < 0.05). Conclusion: MP therapy may help attenuate progressive axon damage, reduce neuronal death, and inhibit microglial/macrophage accumulation, especially when initiated shortly after SCI.